Lead exposure continues to be a major public health problem in the United States. Developing skeletal tissues are targets for Pb, however the molecular mechanisms of its effects have not been defined. The current proposal uses state- of-the-art molecular methods, combining both in vitro and in vivo approaches to investigate the following hypotheses: i) Pb alters the rate at which mesenchymal cells undergo chondrogenesis; ii) Pb alters the rate and organized pattern of chondrocyte differentiation in the growth plate; iii) these effects occur at low concentrations of Pb, consistent with levels observed in Pb-exposed children; and iv) that the effects are mediated by an alteration in specific signaling pathways in the target cells. These hypotheses will be investigated in three aims. In Specific Aim 1, the effect of Pb on chondrogenesis will be examined using mesenchymal cells isolated from stage E11.5 mouse limb buds. Preliminary data demonstrate that Pb enhances the rate of chondrogenesis and the signaling pathways involved in this effect will be identified. Specific Aim 2 investigates the effect of Pb on the rate of chondrocyte differentiation during the process of endochondral bone formation and uses a well-defined embryonic sternal chick chondrocyte cell culture model. This aim is based on strong preliminary data demonstrating important Pb effects on intracellular signaling, growth factor responsiveness, and chondrocyte differentiation. Specific Aim 3 uses an in vivo approach based on an ectopic bone formation model to investigate both chondrogenesis and chondrocyte differentiation in control and Pbexposed mice. This aim is based on preliminary data demonstrating morphological changes in the growth plate of Pb exposed animals as well as abnormal fracture healing in Pb exposed mice. The final aim will characterize the spatial and temporal alterations in gene expression that occur following Pb exposure and which result in abnormal bone formation and skeletal development. Thus, the proposal provides a comprehensive, hypothesis-based examination of the effect of Pb on endochondral bone formation using state-of-the-art molecular methods. The findings have important implications for the management of Pb exposed children and adults in the future.